The invention relates to the Aiolos gene, Aiolos polypeptide, Aiolos homodimers, Aiolos/Ikaros heterodimers and methods of using Aiolos nucleic acids and polypeptides.
In general, the invention features an Aiolos polypeptide, e.g., a polypeptide which includes all or part of the sequence shown in SEQ ID NO:2 or SEQ ID NO:8. The invention also features fragments and analogs of Aiolos polypeptides, preferably having at least one biological activity of an Aiolos polypeptide.
In preferred embodiments, the polypeptide is a recombinant or a substantially pure preparation of an Aiolos polypeptide.
In preferred embodiments, the polypeptide is a vertebrate, e.g., a mammalian, e.g., a human polypeptide.
In preferred embodiments, the Aiolos polypeptide includes additional Aiolos coding sequences 5xe2x80x2 to that of SEQ ID NO:8. In preferred embodiments: the additional sequence includes at least 1, 10, 20, 40, 60, 70, 80 or 100 amino acid residues; the additional sequence is equal to or less than 1, 10, 20, 40, 60, 70, 80 or 100 amino acid residues.
In preferred embodiments: the polypeptide has at least one biological activity, e.g., it reacts with an antibody, or antibody fragment, specific for an Aiolos polypeptide; the polypeptide includes an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:2 or SEQ ID NO:8; the polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:2 or SEQ ID NO:8; the polypeptide is at least 5, 10, 20, 50, 100, 150, 200, or 250 amino acids in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, 150, 200, or 250 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:8; the polypeptide is preferably at least 10, but no more than 100, amino acids in length; the Aiolos polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring Aiolos polypeptide.
In preferred embodiments: the Aiolos polypeptide is encoded by the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with the nucleic acid of SEQ ID NO:1 or SEQ ID NO:7. For example, the Aiolos polypeptide can be encoded by a nucleic acid sequence which differs from a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7 due to degeneracy in the genetic code.
In a preferred embodiment, the Aiolos polypeptide encodes amino acid residues 1-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos polypeptide encodes amino acid residues 58-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos polypeptide encodes amino acid residues 72-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos polypeptide encodes amino acid residues 76-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos polypeptide encodes amino acid residues 1-206 of SEQ ID NO:8.
In a preferred embodiment the Aiolos polypeptide is an agonist of a naturally-occurring mutant or wild type Aiolos polypeptide (e.g., a polypeptide having an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:8). In another preferred embodiment, the polypeptide is an antagonist which, for example, inhibits an undesired activity of a naturally-occurring Aiolos polypeptide (e.g., a mutant polypeptide).
In a preferred embodiment, the Aiolos polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:2 or SEQ ID NO:8. The differences, however, are such that the Aiolos polypeptide exhibits at least one biological activity of an Aiolos polypeptide, e.g., the Aiolos polypeptide retains a biological activity of a naturally occurring Aiolos polypeptide.
In preferred embodiments the Aiolos polypeptide includes an Aiolos polypeptide sequence, as described herein, as well as other N-terminal and/or C-terminal amino acid sequences.
In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:2 or SEQ ID NO:8, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5 to the genomic DNA which encodes a sequence from SEQ ID NO:2 or SEQ ID NO:8.
In yet other preferred embodiments, the Aiolos polypeptide is a recombinant fusion protein having a first Aiolos polypeptide portion and a second polypeptide portion having an amino acid sequence unrelated to an Aiolos polypeptide. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment, the Aiolos polypeptide is a fragment or analog of a naturally occurring Aiolos polypeptide which inhibits reactivity with antibodies, or F(abxe2x80x2)2 fragments, specific for a naturally occurring Aiolos polypeptide.
In a preferred embodiment, the Aiolos polypeptide includes a sequence which is not present in the mature protein.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
In preferred embodiments, the Aiolos polypeptide: is expressed in spleen and thymus; is expressed in mature T and/or B cells; is highly homologous, preferably at least 90% or 95% homologous, with the 50 most C-terminal amino acids of the Ikaros gene (e.g., the dimerization domain of exon 7 of the Ikaros gene); is highly homologous, preferably at least 90% or 95% homologous with the activation domain of exon 7 of the Ikaros gene; is capable of forming Aiolos dimers and/or Aiolos/Ikaros dimers; is involved in lymphocyte differentiation, e.g., T cell maturation.
In preferred embodiments, the Aiolos polypeptide includes: the YAS5 interaction domain; the YAS3 interaction domain; the YIZ Ikaros dimerization domain.
In preferred embodiments, an Aiolos polypeptide encodes: one, two, three, four, five exons, or more exons; exons 3, 4, 5 and 7; exons 3-7; exon 7 (the exons are shown in FIG. 4).
In preferred embodiments, the Aiolos polypeptide has one or more of the following properties:
(a) it can form a dimer with an Aiolos or Ikaros polypeptide;
(b) it is expressed in committed lymphoid progenitors;
(c) it is expressed in committed T and B cells;
(d) it has a molecular weight of approximately 58 kD;
(e) it has at least one zinc finger domain;
(f) it is not expressed in stem cells; or
(g) it is a transcriptional activator of a lymphoid gene.
In other preferred embodiments, the Aiolos polypeptide has one or more of the following properties:
(a) it can form a dimer with an Aiolos or Ikaros polypeptide;
(b) it is expressed in committed lymphoid progenitors;
(c) it is expressed in committed T and B cells;
(d) it has a molecular weight of approximately 58 kD;
(e) it has an N-terminal zinc finger domain;
(f) it is not expressed in stem cells; or
(g) it is a transcriptional activator of a lymphoid gene.
In yet other preferred embodiments, the Aiolos polypeptide has one or more of the following properties:
(a) it can form a dimer with an Aiolos or Ikaros polypeptide;
(b) it is expressed in committed lymphoid progenitors;
(c) it is expressed in committed T and B cells;
(d) it has a molecular weight of approximately 58 kD;
(e) it has at least one or preferably two C-terminal zinc finger domains;
(f) it is not expressed in stem cells; or
(g) it is a transcriptional activator of a lymphoid gene.
The invention includes an immunogen which includes an active or inactive Aiolos polypeptide, or an analog or a fragment thereof, in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the Aiolos polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:2 or SEQ ID NO:8. For example, the immunogen comprises amino acids 1-124 of SEQ ID NO:2 or amino acids 275-448 of SEQ ID NO:2.
The invention also includes an antibody preparation, preferably a monoclonal antibody preparation, specifically reactive with an epitope of the Aiolos immunogen or generally of an Aiolos polypeptide.
In another aspect, the invention provides a substantially pure nucleic acid having, or comprising, a nucleotide sequence which encodes a polypeptide, the amino acid sequence of which includes, or is,.the sequence of an Aiolos polypeptide, or analog or fragment thereof.
In preferred embodiments, the nucleic acid encodes a vertebrate, e.g., a mammalian, e.g., a human polypeptide.
In preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which includes additional Aiolos coding sequences 5xe2x80x2 to that SEQ ID NO:8. In preferred embodiments: the additional sequence includes at least 1, 10, 20, 40, 60, 70, 80 or 100 amino acid residues; the additional sequence is equal to or less than 1, 10, 20, 40, 60, 70, 80 or 100 amino acid residues.
In preferred embodiments, the nucleic acid encodes a polypeptide having one or more of the following characteristics: at least one biological activity of an Aiolos, e.g., a polypeptide specifically reactive with an antibody, or antibody fragment, directed against an Aiolos polypeptide; an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:2 or SEQ ID NO:8; an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:2 or SEQ ID NO:8, the polypeptide is at least 5, 10, 20, 50, 100, 150, 200, or 250 amino acids in length; at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, 150, 200, or 250 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:8; an amino acid sequence which is preferably at least 10, but no more than 100, amino acids in length; the ability to act as an agonist or an antagonist of a biological activity of a naturally occurring Aiolos polypeptide.
In preferred embodiments: the nucleic acid is or includes the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:7; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7; the nucleic acid includes a fragment of SEQ ID NO:1 or SEQ ID NO:7 which is at least 25, 50, 100, 200, 300, 400, 500, or 1,000 bases in length; the nucleic acid differs from the nucleotide sequence of SEQ ID NO:1 due to degeneracy in the genetic code.
In a preferred embodiment, the Aiolos encoding nucleic acid sequence encodes amino acid residues 1-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos encoding nucleic acid sequence encodes amino acid residues 58-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos encoding nucleic acid sequence encodes amino acid residues 72-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos encoding nucleic acid sequence encodes amino acid residues 76-507 of SEQ ID NO:2 or a functionally equivalent residue in the Aiolos sequence of another vertebrate or mammal, e.g., a human.
In a preferred embodiment, the Aiolos encoding nucleic acid sequence encodes amino acid residues 1-206 of SEQ ID NO:8.
In a preferred embodiment the polypeptide encoded by the nucleic acid is an agonist which, for example, is capable of enhancing an activity of a naturally-occurring mutant or wild type Aiolos polypeptide. In another preferred embodiment, the encoded polypeptide is an antagonist which, for example, inhibits an undesired activity of a naturally-occurring Aiolos polypeptide (e.g., a polypeptide having an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:8).
In a preferred embodiment, the encoded Aiolos polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:2 or SEQ ID NO:8. The differences, however, are such that the encoded Aiolos polypeptide exhibits at least one biological activity of a naturally occurring Aiolos polypeptide (e.g., the Aiolos polypeptide of SEQ ID NO:2 or SEQ ID NO:8).
In preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which includes an Aiolos polypeptide sequence, as described herein, as well as other N-terminal and/or C-terminal amino acid sequences.
In preferred embodiments, the nucleic acid encodes a polypeptide which includes all or a portion of an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:8, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:2 or SEQ ID NO:8.
In preferred embodiments, the encoded polypeptide is a recombinant fusion protein having a first Aiolos polypeptide portion and a second polypeptide portion having an amino acid sequence unrelated to an Aiolos polypeptide. The second polypeptide portion can be, e.g., any of glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain. In preferred embodiments the fusion protein can be used in a two-hybrid assay.
In preferred embodiments, the encoded polypeptide is a fragment or analog of a naturally occurring Aiolos polypeptide which inhibits reactivity with antibodies, or F(abxe2x80x2)2 fragments, specific for a naturally occurring Aiolos polypeptide.
In preferred embodiments, the nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Aiolos gene sequence, e.g., to render the Aiolos gene sequence suitable for use as an expression vector.
In yet another preferred embodiment, the nucleic acid of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from SEQ ID NO:1 or SEQ ID NO:7, or more preferably to at least 20 consecutive nucleotides from SEQ ID NO:1 or SEQ ID NO:7, or more preferably to at least 40 consecutive nucleotides from SEQ ID NO:1 or SEQ ID NO:7.
In a preferred embodiment, the nucleic acid encodes an Aiolos polypeptide which includes a sequence which is not present in the mature protein.
In preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which: is expressed in spleen and thymus; is expressed in mature T and/or B cells; is highly homologous, preferably at least 90% or 95% homologous, with the 50 most C-terminal amino acids of the Ikaros gene (e.g., the dimerization domain of exon 7 of the Ikaros gene); is highly homologous, preferably at least 90% or 95% homologous, with the activation domain of exon 7 of the Ikaros gene; is capable of forming Aiolos dimers and/or Aiolos/Ikaros dimers; is involved in lymphocyte differentiation, e.g., T cell maturation.
In preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which includes: the YAS5 interaction domain; the YAS3 interaction domain; the YIZ Ikaros dimerization domain.
In preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which encodes: one, two, three, four, five exons, or more exons; exons 3, 4, 5 and 7; exons 3-7; exon 7 (the exons are shown in FIG. 4).
In preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which has one or more of the following properties:
(a) it can form a dimer with an Aiolos or Ikaros polypeptide;
(b) it is expressed in committed lymphoid progenitors;
(c) it is expressed in committed T and B cells;
(d) it has a molecular weight of approximately 58 kD;
(e) it has at least one zinc finger domain;
(f) it is not expressed in stem cells; or
(g) it is a transcriptional activator of a lymphoid gene.
In other preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which has one or more of the following properties:
(a) it can form a dimer with an Aiolos or Ikaros polypeptide;
(b) it is expressed in committed lymphoid progenitors;
(c) it is expressed in committed T and B cells;
(d) it has a molecular weight of approximately 58 kD;
(e) it has an N-terminal zinc finger domain;
(f) it is not expressed in stem cells; or
(g) it is a transcriptional activator of a lymphoid gene.
In yet other preferred embodiments, the nucleic acid encodes an Aiolos polypeptide which has one or more of the following properties:
(a) it can form a dimer with an Aiolos or Ikaros polypeptide;
(b) it is expressed in committed lymphoid progenitors;
(c) it is expressed in committed T and B cells;
(d) it has a molecular weight of approximately 58 kD;
(e) it has at least one or preferably two C-terminal zinc finger domains;
(f) it is not expressed in stem cells; or
(g) it is a transcriptional activator of a lymphoid gene.
In another aspect, the invention includes: a vector including a nucleic acid which encodes an Aiolos polypeptide; a host cell transfected with the vector; and a method of producing a recombinant Aiolos polypeptide, including culturing the cell, e.g., in a cell culture medium, and isolating the Aiolos polypeptide, e.g., an Aiolos polypeptide from the cell or from the cell culture medium.
In another aspect, the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:8.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO:1 or SEQ ID NO:8, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
The invention includes vertebrate, e.g., mammalian, e.g., rodent, e.g., mouse or rat, or human Aiolos polypeptides.
In another aspect, the invention features a method of evaluating a compound for the ability to interact with, e.g., bind, or modulate, e.g., inhibit or promote, the activity of an Aiolos polypeptide, e.g., an Aiolos monomer, or an Aiolos-Aiolos dimer or an Aiolos-Ikaros dimer. The method includes contacting the compound with the Aiolos polypeptide, and evaluating the ability of the compound to interact with or form a complex with the Aiolos polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with the Aiolos polypeptide. It can also be used to find natural or synthetic inhibitors of mutant or wild type Aiolos polypeptide. The compound can be a peptide or a non peptide molecule, e.g., a small molecule preferably 500 to 5,000 molecular weight, more preferably 500 to 1,000 molecular weight, having an aromatic scaffold, e.g., a bis-amide phenol, decorated with various functional groups.
In brief, a two hybrid assay system (see e.g., Bartel et al. (1993) Cellular Interaction, in Development: A practical Approach, D. A. Hartley, ed., Oxford University Press, Oxford, pp. 153-179) allows for detection of protein-protein interactions in yeast cells. The known protein, e.g., an Aiolos polypeptide, is often referred to as the xe2x80x9cbaitxe2x80x9d protein. The proteins tested for binding to the bait protein are often referred to as xe2x80x9cfishxe2x80x9d proteins. The xe2x80x9cbaitxe2x80x9d protein, e.g., an Aiolos polypeptide, is fused to the GAL4 DNA binding domain. Potential xe2x80x9cfishxe2x80x9d proteins are fused to the GAL4 activating domain. If the xe2x80x9cbaitxe2x80x9d protein and a xe2x80x9cfishxe2x80x9d protein interact, the two GAL4 domains are brought into close proximity, thus rendering the host yeast cell capable of surviving a specific growth selection.
In another aspect, the invention features a method of identifying active fragments or analogs of an Aiolos polypeptide. The method includes first identifying a compound, e.g., an Ikaros peptide, which interacts with an Aiolos polypeptide and determining the ability of the compound to bind the candidate fragment or analog. The two hybrid assay described above can be used to obtain fragment-binding compounds. These compounds can then be used as xe2x80x9cbaitxe2x80x9d to fish for and identify fragments of the Aiolos polypeptide which interact, bind, or form a complex with these compounds.
In another aspect, the invention features a method of making an Aiolos polypeptide, having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring Aiolos polypeptide. The method includes altering the sequence of an Aiolos polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:8) by, for example, substitution or deletion of one or more residues of a non-conserved region, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features a method of making a fragment or analog of an Aiolos polypeptide, e.g., an Aiolos polypeptide having at least one biological activity of a naturally occurring Aiolos polypeptide. The method includes altering the sequence, e.g., by substitution or deletion of one or more residues, preferably which are non-conserved residues, of an Aiolos polypeptide, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding an Aiolos gene regulatory sequence. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid. In preferred embodiments the Aiolos gene regulatory sequence is functionally linked to a heterologous gene, e.g., a reporter gene.
In another aspect, the invention features a human cell, e.g., a hematopoietic stem cell or a lymphocyte e.g., a T or a B cell, transformed with a nucleic acid which encodes an Aiolos polypeptide.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder, e.g., an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, including administering a therapeutically-effective amount of an Aiolos polypeptide to the animal. The Aiolos polypeptide can be monomeric or an Aiolos-Aiolos or Aiolos-Ikaros dimer.
In preferred embodiments: the disorder is characterized by unwanted, e.g., higher than normal, antibody, e.g., IgE, production or levels; the disorder is characterized by an antibody mediated response, e.g., an IgE mediated response; the disorder is characterized by an aberrant or unwanted B cell response; the disorder is asthma, an immune mediated skin disorder, e.g., excema, an allergic reaction, hay fever, hives, a food allergy; the disorder is characterized by a hypersensitive response, e.g., an IgE mediated hypersensitive response; the disorder is characterized by an anaphylactic response; the disorder is characterized by a local B cell mediated response; the disorder is characterized by a systemic B cell mediated response; the disorder is characterized by unwanted mast cell degranulation.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse. The method includes administering to the animal a cell selected, e.g., selected in vitro, for the expression of a product of the Aiolos gene, e.g., hematopoietic stem cells, e.g., cells transformed with Aiolos-peptide-encoding DNA, e.g., hematopoietic stem cells transformed with Aiolos-peptide-encoding DNA.
In preferred embodiments: the disorder is characterized by unwanted, e.g., higher than normal, antibody, e.g., IgE, production or levels; the disorder is characterized by an antibody mediated response, e.g., an IgE mediated response; the disorder is characterized by an aberrant or unwanted B cell response; the disorder is asthma,.an immune mediated skin disorder, e.g., excema, an allergic reaction, hay fever, hives, a food allergy; the disorder is characterized by a hypersensitive response, e.g., an IgE mediated hypersensitive response; the disorder is characterized by an anaphylactic response; the disorder is characterized by a local B cell mediated response; the disorder is characterized by a systemic B cell mediated response; the disorder is characterized by unwanted mast cell degranulation.
In preferred embodiments: the cells are taken from the animal to which they are administered; the cells are taken from an animal which is MHC matched with the animal to which they are administered; the cells are taken from an animal which is syngeneic with the animal to which they are administered; the cells are taken from an animal which is of the same species as is the animal to which they are administered.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse. The method includes administering to the animal a nucleic acid encoding an Aiolos peptide and expressing the nucleic acid.
In preferred embodiments: the disorder is characterized by unwanted, e.g., higher than normal, antibody, e.g., IgE, production or levels; the disorder is characterized by an antibody mediated response, e.g., an IgE mediated response; the disorder is characterized by an aberrant or unwanted B cell response; the disorder is asthma, an immune mediated skin disorder, e.g., excema, an allergic reaction, hay fever, hives, a food allergy; the disorder is characterized by a hypersensitive response, e.g., an IgE mediated hypersensitive response; the disorder is characterized by an anaphylactic response; the disorder is characterized by a local B cell mediated response; the disorder is characterized by a systemic B cell mediated response; the disorder is characterized by unwanted mast cell degranulation.
In another aspect, the invention features a method of evaluating the effect of a treatment, e.g., a treatment designed to promote or inhibit hematopoiesis, including carrying out the treatment and evaluating the effect of the treatment on the expression of the Aiolos gene.
In preferred embodiments the treatment is administered: to an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, or a cell, e.g., a cultured stem cell.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Aiolos gene, e.g., a proliferative disorder, e.g., a leukemic disorder, Hodgkin""s lymphoma, a cutaneuous cell lymphoma, e.g., a cutaneous T cell lymphoma, or a disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells. The method includes examining the subject for the expression of the Aiolos gene, non-wild type expression or mis-expression being indicative of risk.
In preferred embodiments: the disorder is characterized by unwanted, e.g., higher than normal, antibody, e.g., IgE, production or levels; the disorder is characterized by an antibody mediated response, e.g., an IgE mediated response; the disorder is characterized by an aberrant or unwanted B cell response; the disorder is asthma, an immune mediated skin disorder, e.g., excema, an allergic reaction, hay fever, hives, a food allergy; the disorder is characterized by a hypersensitive response, e.g., an IgE mediated hypersensitive response; the disorder is characterized by an anaphylactic response; the disorder is characterized by a local B cell mediated response; the disorder is characterized by a systemic B cell mediated response; the disorder is characterized by unwanted mast cell degranulation.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Aiolos gene, e.g., a a proliferative disorder, e.g., a leukemic disorder, Hodgkin""s lymphoma, a cutaneuous cell lymphoma, e.g., a cutaneous T cell lymphoma, or a disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells. The method includes providing a nucleic acid sample from the subject and determining if the structure of an Aiolos gene allele of the subject differs from wild type.
In preferred embodiments: the disorder is characterized by unwanted, e.g., higher than normal, antibody, e.g., IgE, production or levels; the disorder is characterized by an antibody mediated response, e.g., an IgE mediated response; the disorder is characterized by an aberrant or unwanted B cell response; the disorder is asthma, an immune mediated skin disorder, e.g., excema, an allergic reaction, hay fever, hives, a food allergy; the disorder is characterized by a hypersensitive response, e.g., an IgE mediated hypersensitive response; the disorder is characterized by an anaphylactic response; the disorder is characterized by a local B cell mediated response; the disorder is characterized by a systemic B cell mediated response; the disorder is characterized by unwanted mast cell degranulation.
In preferred embodiments: the determination includes determining if an Aiolos gene allele of the subject has a gross chromosomal rearrangement; the determination includes sequencing the subject""s Aiolos gene.
In another aspect, the invention features, a method of evaluating an animal or cell model for a a proliferative disorder, e.g., a leukemic disorder, Hodgkin""s lymphoma, a cutaneuous cell lymphoma, e.g., a cutaneous T cell lymphoma, or an immune disorder, e.g., a T cell related disorder, e.g., a disorder characterized by a shortage of T or B cells. The method includes determining if the Aiolos gene in the animal or cell model is expressed at a predetermined level or if the Aiolos gene is mis-expressed. In preferred embodiments: the predetermined level is lower than the level in a wild type or normal animal; the predetermined level is higher than the level in a wild type or normal animal; or the pattern of isoform expression is altered from wildtype.
In preferred embodiments: the disorder is characterized by unwanted, e.g., higher than normal, antibody, e.g., IgE, production or levels; the disorder is characterized by an antibody mediated response, e.g., an IgE mediated response; the disorder is characterized by an aberrant or unwanted B cell response; the disorder is asthma, an immune mediated skin disorder, e.g., excema, an allergic reaction, hay fever, hives, a food allergy; the disorder is characterized by a hypersensitive response, e.g., an IgE mediated hypersensitive response; the disorder is characterized by an anaphylactic response; the disorder is characterized by a local B cell mediated response; the disorder is characterized by a systemic B cell mediated response; the disorder is characterized by unwanted mast cell degranulation.
In another aspect, the invention features, a transgenic animal, e.g., a mammal, e.g., a mouse or a nonhuman primate having an Aiolos transgene.
In preferred embodiments the animal is a transgenic mouse having a mutated Aiolos transgene, the mutation occurring in, or altering, e.g., a domain of the Aiolos gene described herein.
In other preferred embodiments the transgenic animal or cell: is heterozygous for an Aiolos transgene; homozygous for an Aiolos transgene; includes a first Aiolos transgene and a second Aiolos transgene; includes an Aiolos transgene and a second transgene which is other than an Aiolos transgene, e.g., an Ikaros transgene.
In another aspect, the invention features a method for evaluating the effect of a treatment on a transgenic cell or animal having an Aiolos transgene, e.g., the effect of the treatment on the development of the immune system. The method includes administering the treatment to a cell or animal having an Aiolos transgene, and evaluating the effect of the treatment on the cell or animal. The effect can be, e.g., the effect of the treatment on: Aiolos or Ikaros expression or misexpression; the immune system or a component thereof; the nervous system or a component thereof; or the cell cycle. Immune system effects include e.g., T cell activation, T cell development, the ability to mount an immune response, the ability to give rise to a component of the immune system, B cell development, NK cell development, or the ratios CD4+/CD8+, CD4+/CD8xe2x88x92 and CD4xe2x88x92/CD8+.
In preferred embodiments the treatment can include: the administration of a drug, chemical, or other substance; the administration of ionizing radiation; the administration of an antibody, e.g., an antibody directed against a molecule or cell of the immune system; administration of a substance or other treatment which suppresses the immune system; or administration of a substance or other treatment which activates or boosts the function of the immune system; introduction of a nucleic acid, e.g., a nucleic acid which encodes or expresses a gene product, e.g., a component of the immune system; the introduction of a protein, e.g., a protein which is a component of the immune system.
In another aspect, the invention features a method for evaluating the effect of a treatment on an immune system component. The method includes: (1) supplying a transgenic cell or animal having an Aiolos transgene; (2) supplying the immune system component; (3) administering the treatment; and (4) evaluating the effect of the treatment on the immune system component.
In yet another aspect, the invention features a method for evaluating the interaction of a first immune system component with a second immune system component. The method includes: (1) supplying a transgenic cell or animal, e.g., a mammal, having an Aiolos transgene; (2) introducing the first and second immune system component into the transgenic cell or mammal; and (3) evaluating an interaction between the first and second immune system components.
Mice with mutant Aiolos transgenes which eliminate many of the normal components of the immune system, e.g., mice homozygous for a transgene having a deletion for some or all of exon 7 (corresponding to amino acids 275-507 of SEQ ID NO:2), are particularly useful for xe2x80x9creconstitution experiments.xe2x80x9dIn another aspect, the invention features a method for evaluating the effect of a treatment on an immune system disorder, e.g., a neoplastic disorder, a leukemia or a lymphoma, a T cell related lymphoma, including: administering the treatment to a cell or animal having an Aiolos transgene, and evaluating the effect of the treatment on the cell or animal. The effect can be, e.g., the effect of the treatment on: Aiolos or Ikaros expression or misexpression; the immune system or a component thereof; or the cell cycle. Immune system effects include e.g., T cell activation, T cell development, the ability to mount an immune response, the ability to give rise to a component of the immune system, B cell development, NK cell development, or the ratios CD4+/CD8+, CD4+/CD8xe2x88x92 and CD4xe2x88x92/CD8+.
The inventors have also discovered that Ikaros and Aiolos can form dimers (heterodimers) with other polypeptides. E.g., an Ikaros polypeptide can form dimers not only with Ikaros polypeptides, but with other polypeptides which bind to its C terminal region, e.g, other polypeptides having Zinc-finger regions, e.g., Aiolos polypeptides. Similarly, an Aiolos polypeptide can form dimers not only with Aiolos polypeptides, but with other polypeptides which bind to its C terminal region, e.g, other polypeptides having Zinc-finger regions, e.g., Ikaros polypeptides.
The invention also includes Ikaros-Aiolos dimers. The Ikaros member of the dimer can be any Ikaros polypeptide, e.g., any naturally occuring Ikaros or any Ikaros referred to in U.S. Ser. No. 08/238,212, filed May 2, 1994, hereby incorporated by reference. The proteins of the Ikaros family are isoforms which arise from differential splicing of Ikaros gene transcripts. The isoforms of the Ikaros family generally include a common 3xe2x80x2 exon (Ikaros exon E7, which includes amino acid residues 283-518 of the mouse Ikaros protein represented by SEQ ID NO:18, and amino acid residues 229-461 of the human Ikaros protein represented by SEQ ID NO:16) but differ in the 5xe2x80x2 region. The Ikaros family includes all naturally occurring splicing variants which arise from transcription and processing of the Ikaros gene. Five such isoforms are described herein and in U.S. Ser. No. 08/238,212, filed May 2, 1994, hereby incorporated by reference. The Ikaros family also includes other isoforms, including those generated by mutagenesis and/or by in vitro exon shuffling. The naturally occurring Ikaros proteins can bind and activate (to differing extents) the enhancer of the CD3 xcex4 gene, and are expressed primarily in early hematopoietic and lymphoid cells in the adult. The expression pattern of this transcription factor during embryonic development suggests that Ikaros proteins play a role as a genetic switch regulating entry into the lymphoid and T cell lineages. The Ikaros gene is also expressed in the proximal corpus striatum during early embryogenesis in mice. As is discussed herein, Ikaros and Aiolos polypeptide can form Ikaros-Aiolos dimers.
Accordingly, the invention includes a substantially pure dimer which includes (or consiststs essentially of) an Aiolos polypeptide and an Ikaros polypeptide.
The Ikaros polypeptide of the Ikaros-Aiolos dimer includes one or more Ikaros exons. In preferred embodiments: the Ikaros exon is E1/2, E3, E4, E5, E6, or E7; the peptide does not include exon E7.
In other preferred embodiments: the Ikaros peptide of the Ikaros-Aiolos dimer further includes a second Ikaros exon; the second exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7 and the second exon is any of E1/2, E3, E4, E5, E6.
In other preferred embodiments: the Ikaros peptide of the Ikaros-Aiolos dimer further includes a third Ikaros exon; the third exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E3, and the third exon is E1/2; the peptide is Ikaros isoform 5.
In other preferred embodiments: the Ikaros peptide of the Ikaros-Aiolos dimer further includes a fourth Ikaros exon; the fourth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E4, the third exon is E3, and the fourth exon is E1/2; the first exon is E7, the second exon is E4, the third exon is E3, and the fourth exon is E1/2; the peptide is Ikaros isoform 3 or 4.
In other preferred embodiments: the Ikaros peptide of the Ikaros-Aiolos dimer further includes a fifth Ikaros exon; the fifth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, and the fifth exon is E1/2; the peptide is Ikaros Isoform 2.
In other preferred embodiments: the Ikaros peptide of the Ikaros-Aiolos dimer further includes a sixth Ikaros exon; the sixth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, the fifth exon is E3, and the sixth exon is E1/2; the peptide is Ikaros isoform 1. In preferred embodiments: the sequence of the Ikaros exon is essentially the same as that of a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the amino acid sequence of the Ikaros exon is such that a nucleic acid sequence which encodes it is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., Ikaros having an amino acid sequence represented in any of SEQ ID NOS:15-21 or SEQ ID NO:22; the amino acid sequence of the Ikaros exon is such that a nucleic acid sequence which encodes it hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon with the same, or essentially the same, amino acid sequence as an Ikaros exon represented in any of SEQ ID NOS: 15-21 the amino acid sequence of the Ikaros exon is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the encoded Ikaros amino acid sequence is at least 50% more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the Ikaros exon is essentially equal in length to a naturally occurring Ikaros exon; the amino acid sequence of the Ikaros exon is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with a naturally occurring Ikaros exon sequence, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21; the Ikaros exon amino acid sequence is the same, or essentially the same, as that of a naturally occurring Ikaros exon, or a fragment of the sequence thereof, e.g., an Ikaros exon described in any of SEQ ID NOS: 15-21; and the peptide has Ikaros peptide activity; the peptide has Ikaros antagonist activity.
In preferred embodiments: the Ikaros protein of the Ikaros-Aiolos dimer comprises a polypeptide represented by the general formula A-B-C-D-E, wherein A represents Exon 3 or is absent, B represents Exon 4 or is absent, C represents Exon 5 or is absent, D represents Exon 6 or is absent, and E represents Exon 7 or is absent; the polypeptide includes at least two of said exons; the polypeptide includes at least one exon containing a zinc finger domain; the polypeptide includes at least one exon selected from E3, E4 or E5.
In preferred embodiments: the exons in the Ikaros peptide of the Ikaros-Aiolos dimer are arranged in the same relative linear order as found in a naturally occurring isoform, e.g., in Ikaros isoform 1, e.g., in a peptide having the exons E3 and E7, E3 is located N-terminal to E7; the linear order of the exons is different from that found in a naturally occurring isoform, e.g., in Ikaros isoform 1, e.g., in a peptide having exons E3, E5, and E7, the direction N-terminal to C-terminal end, is E5, E3, E7; the exons in the peptide differ in one or more of composition (i.e., which exons are present), linear order, or number (i.e., how many exons are present or how many times a given exon is present) from a naturally occurring Ikaros isoform, e.g., from Ikaros isoform 1, 2, 3, 4, or 5; e.g. the Ikaros protein is an isoform generated by in vitro exon shuffling.
The invention also includes: a cell, e.g., a cultured cell or a stem cell, containing purified Ikaros-protein-encoding-DNA and purified Aiolos-protein-encoding -DNA; a cell capable of expressing an Ikaros and an Aiolos protein; a cell capable of giving rise to a transgenic animal or to a homogeneous population of hemopoietic cells, e.g., lymphoid cells, e.g., T cells; an essentially homogeneous population of cells, each of which includes purified Ikaros-protein-encoding-DNA and purified Aiolos-protein-encoding -DNA ; and a method for manufacture of a dimer of the invention including culturing a cell which includes a DNA, preferably a purified DNA, of the invention in a medium to express the peptides.
The invention also includes: a preparation of cells, e.g., cultured cells or a stem cells, including a cell a containing purified Ikaros-protein-encoding-DNA and a cell encoding purified Aiolos-protein-encoding -DNA.
The invention also includes substantially pure preparation of an antibody, preferably a monoclonal antibody directed against an Ikaros-Aiolos dimer (which preferably does not bind to an Ikaros-Ikaros or Aiolos-Aiolos dimer); a therapeutic composition including an Ikaros-Aiolos dimer and a pharmaceutically acceptable carrier; a therapeutic composition which includes a purified DNA of the invention and a pharmaceutically acceptable carrier.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, including administering a therapeutically-effective amount of an Ikaros-Aiolos dimer to the animal.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse including administering to the animal cells selected, e.g., selected in vitro, for the expression of a product of the Ikaros gene and of the Aiolos gene, e.g., hematopoietic stem cells, e.g., cells transformed with Ikaros-peptide-encoding DNA and or Aiolos-peptide-encoding DNA, e.g., hematopoietic stem cells transformed with Ikaros and or Aiolos-peptide-encoding DNA. The Ikaros and Aiolos DNA can be present in the same or in different cells.
In preferred embodiments: the cells are taken from the animal to which they are administered; the cells are taken from an animal which is MHC matched with the animal to which they are administered; the cells are taken from an animal which is syngeneic with the animal to which they are administered; the cells are taken from an animal which is of the same species as is the animal to which they are administered.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, including administering to the animal a nucleic acid encoding an Ikaros peptide and a nucleic acid encoding an Aiolos peptide and expressing the nucleic acids.
In another aspect, the invention features a method of evaluating the effect of a treatment, e.g., a treatment designed to promote or inhibit hematopoiesis, including carrying out the treatment and evaluating the effect of the treatment on the expression of the Ikaros and the Aiolos gene.
In preferred embodiments the treatment is administered: to an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, or a cell, e.g., a cultured stem cell.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Ikaros gene, e.g., a leukemic disorder or other disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells, including examining the subject for the expression of the Ikaros-Aiolos dimers, non-wild type expression or mis-expression being indicative of risk.
In another aspect, the invention features, a method of evaluating an animal or cell model for an immune disorder, e.g., a T cell related disorder, e.g., a disorder characterized by a shortage of T or B cells, including determining if Ikaros-Aiolos dimers in the animal or cell model are expressed at a predetermined level. In preferred embodiments: the predetermined level is lower than the level in a wild type or normal animal; the predetermined level is higher than the level in a wild type or normal animal; or the pattern of isoform expression is altered from wildtype.
In another aspect, the invention features a transgenic rodent, e.g., a mouse, having a transgene which includes an Ikaros gene or Ikaros protein encoding DNA and an Aiolos gene or Aiolos protein encoding DNA. In preferred embodiments: the Ikaros and or Aiolos gene or DNA includes a deletion, e.g. a deletion of all or part of one or more exons.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, immune system disorder, including administering a therapeutically effective amount of an Ikaros-Aiolos dimer to the animal.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including administering to the animal cells selected, e.g., selected in vitro, for the production of an Ikaros-Aiolos dimer, e.g., hematopoietic stem cells, e.g., cells transformed with Ikaros and or Aiolos protein-encoding DNA, e.g., hematopoietic stem cells transformed with Ikaros and or Aiolos-protein-encoding DNA.
In preferred embodiments: the cells are taken from the animal to which they are administered; the cells are taken from an animal which is MHC matched with the animal to which they are administered; the cells are taken from an animal which is syngeneic with the animal to which they are administered: the cells are taken from an animal which is of the same species as is the animal to which they are administered.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including administering to the animal a nucleic acid encoding an Ikaros peptide and a nucleic acid encoding an Aiolos peptide and expressing the nucleic acids.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of an Ikaros-Aiolos dimer, e.g., a disorder of the nervous system, e.g., a disorder of the corpus striatum, e.g., Alzheimer""s disease, including examining the subject for the expression of an Ikaros-Aiolos dimer, non-wild type expression or mis-expression being indicative of risk.
In another aspect, the invention features, a method of inhibiting an interaction, e.g., binding, between a protein, e.g., an Ikaros isoform, Aiolos, an Ikaros-Ikaros dimer, an Aiolos-Aiolos dimer, or a first Ikaros-Aiolos dimer, and a DNA sequence, e.g., a DNA sequence under the control of a xcex4A sequence, an NKFB sequence, a sequence which corresponds to an Ikaros or Aiolos binding site, or a site present in the control region of a lymphocyte restricted gene, e.g., TCR-xcex1, -xcex2, or -xcex4, CD3 xcex4, -xcex5, -xcex3 genes, the SL3 gene, or the HIV LTR gene. The methods includes contacting the DNA sequence with an effective amount of a second Ikaros-Aiolos dimer, e.g., an Ikaros-Aiolos dimer described herein.
In another aspect, the invention features, a method of inhibiting an interaction, e.g., binding, between a protein, e.g., an Ikaros isoform, Aiolos, an Ikaros-Ikaros dimer, an Aiolos-Aiolos dimer, or an Ikaros-Aiolos dimer, and a DNA sequence, e.g., a xcex4A sequence, an NKFB sequence, a sequence which corresponds to an Ikaros binding oligonucleotide described herein, or a site present in the control region of a lymphocyte restricted gene, e.g., TCR-xcex5, -xcex3, or -xcex4, CD3 xcex4, -xcex5, -xcex3 genes, the SL3 gene, or the HIV LTR gene. The methods includes contacting the protein with an effective amount of an Ikaros, Aiolos, or Ikaros-Aiolos dimer-binding oligonucleotide.
In another aspect, the invention features, a method of modulating hematopoietic development, e.g., a progression of a cell through a lymphoid lineage, e.g., a lymphocyte maturation and/or function, the method including altering, in a cell or animal, a wild type expression of Ikaros-Aiolos and/or Aiolos-Aiolos dimers.
In preferred embodiments, the expression can be altered by providing Aiolos and/or Ikaros polypeptides.
In other preferred embodiments, the method includes supplying to a cell or animal a mutant Aiolos and/or Ikaros polypeptide, e.g., a polypeptide having a dominant negative mutation, e.g., a DNA binding mutation.
In another aspect, the invention features, a method of modulating hematopoietic development, e.g., a progression of a cell through a lymphoid lineage, e.g., a lymphocyte maturation and/or function, the method including altering, in a cell or animal, the ratio of Ikaros-Ikaros dimers to any of Aiolos-Aiolos or Aiolos-Ikaros dimers.
In preferred embodiments, the ratio can be altered by providing Aiolos or Ikaros polypeptides.
In other preferred embodiments, the method includes supplying to a cell or animal a mutant Aiolos and/or Ikaros polypeptide, e.g., a polypeptide having a dominant negative mutation, e.g., a DNA binding mutation.
In another aspect, the invention features, a method of modulating hematopoietic development, e.g., a progression of a cell through a lymphoid lineage, e.g., a lymphocyte maturation and/or function, the method including altering, in a cell or animal, the ratio of Aiolos-Aiolos dimers to any of Ikaros-Ikaros or Aiolos-Ikaros dimers.
In preferred embodiments, the ratio can be altered by providing Aiolos or Ikaros polypeptides.
In other preferred embodiments, the method includes supplying to a cell or animal a mutant Aiolos and/or Ikaros polypeptide, e.g., a polypeptide having a dominant negative mutation, e.g., a DNA binding mutation.
In general, the invention features, a method of providing a proliferation-deregulated cell, or a cell which has non-wild type, e.g., increased, antibody production. The method includes: providing a mammal having a cell which misexpresses Aiolos, e.g., a hematopoietic cell; and isolating a proliferation-deregulated or antibody overexpressing cell from the mammal. The proliferation-deregulated or antibody overexpressing cell can be, e.g., a hematopoietic cell, e.g., a B lymphocyte.
In preferred embodiments: the mammal is a non-human mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse.
In a preferred embodiment, the method further includes: allowing the Aiolos-misexpressing cell to divide and give rise to a proliferation-deregulated or antibody producing cell, e.g., a lymphocyte; providing a plurality of the proliferation-deregulated cells e.g., lymphocytes or transformed lymphocytes from the mammal.
In preferred embodiments: the proliferation-deregulated or antibody producing cell e.g., a lymphocyte, e.g., a transformed lymphocyte, is isolated from a lymphoma of the mammal.
In preferred embodiments: the mammal is heterozygous at the Aiolos locus; the mammal carries a mutation at the Aiolos gene, e.g., a point mutation in or a deletion for all or part of the Aiolos gene, e.g., a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediates DNA binding of the Aiolos protein or for one or more of the two C terminal zinc finger regions which mediate dimerization of the Aiolos protein; the mammal is heterozygous. or homozygous for an Aiolos transgene; the mammal carries a mutation in the control region of the Aiolos gene.
In preferred embodiments: the mammal carries a mutation at the Aiolos gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the mammal carries deletion for all or part of exon 7.
In preferred embodiments: the proliferation-deregulated or antibody producing cell is a homozygous mutant Aiolos cell e.g., a lymphocyte; the proliferation-deregulated or antibody producing lymphocyte is a B lymphocyte; the proliferation-deregulated or antibody producing cell is heterozygous or homozygous for an Aiolos transgene.
In preferred embodiments, the cell is a lymphocyte and is: a cell which secretes one or more anti-inflammatory cytokines; a cell which is antigen or idiotype specific; a cell which produces, or over produces, antibodies, e.g., IgG, IgA, or IgE antibodies.
In a preferred embodiment: the Aiolos-misexpressing cell, e.g., a lymphocyte, is supplied exogenously to the mammal, e.g., to a homozygous wild-type Aiolos mammal or a mammal carrying a mutation at the Aiolos gene, e.g., a point mutation or a deletion for all or part of the Aiolos gene. If exogenously supplied, the cell can be a human or a nonhuman, e.g., a swine, nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, lymphocyte.
In a preferred embodiment the method further comprises isolating one or more cells, e.g., lymphocytes, from the mammal, and allowing the cell or cells to proliferate into a clonal population of cells, e.g., lymphocytes.
In preferred embodiments: the mammal is immunized with an antigen; the cell is exogenously supplied and one or both of the mammal or the mammal which donates the cell are immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen; an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In preferred embodiments the method further includes providing a lymphocyte e.g., a B lymphocyte, or a substantially homogenous population of lymphocytes, e.g,. B lymphocytes, which produce an antibody molecule, e.g. an IgG, IgA, or IgE molecule, which recognizes a selected antigen.
In another aspect, the invention features, a method of providing a proliferation-deregulated cell, or a cell which has non-wild type, e.g., increased, antibody production. The method includes: causing a subject cell to misexpress the Aiolos gene, e.g., by inducing an Aiolos mutation, thereby providing a a proliferation-deregulated or antibody overexpressing cell. The proliferation-deregulated or antibody overexpressing cell can be, e.g., a hematopoietic cell, e.g., a B lymphocyte.
In preferred embodiments: the subject cell is from a non-human mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse.
In a preferred embodiment, the method further includes: allowing the Aiolos-misexpressing cell to divide and give rise to a proliferation-deregulated or antibody producing cell, e.g., a lymphocyte; providing a plurality of the proliferation-deregulated cells e.g., lymphocytes or transformed lymphocytes from the mammal.
In preferred embodiments: the proliferation-deregulated or antibody producing cell e.g., a lymphocyte, e.g., a transformed lymphocyte, is isolated from cell or tissue culture.
In preferred embodiments: the cell is heterozygous at the Aiolos locus; the cell carries mutation at the Aiolos gene, e.g., a point mutation in or a deletion for all or part of the Aiolos gene, e.g., a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediates DNA binding of the Aiolos protein or for one or more of the two C terminal zinc finger regions which mediate dimerization of the Aiolos protein; the mammal is heterozygous or homozygous for an Aiolos transgene; the cell carries a mutation in the control region of the Aiolos gene.
In preferred embodiments: the cell carries a mutation at the Aiolos gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the mammal carries deletion for all or part of exon 7.
In preferred embodiments: the proliferation-deregulated or antibody producing cell is a homozygous mutant Aiolos cell e.g., a lymphocyte; the proliferation-deregulated or antibody producing lymphocyte is a B lymphocyte; the proliferation-deregulated or antibody producing cell is heterozygous or homozygous for an Aiolos transgene.
In preferred embodiments, the cell is a lymphocyte and is: a cell which secretes one or more anti-inflammatory cytokines; a cell which is antigen or idiotype specific; a cell which produces, or over produces, antibodies, e.g., IgG, IgA, or IgE antibodies.
In a preferred embodiment the method further comprises allowing the subject cell, to proliferate into a clonal population of cells, e.g., lymphocytes.
In preferred embodiments: the mammal which supplies the subject cell is immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen; an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In preferred embodiments the method further includes providing a lymphocyte e.g., a B lymphocyte, or a substantially homogenous population of lymphocytes, e.g,. B lymphocytes, which produce an antibody molecule, e.g. an IgG, IgA, or IgE molecule, which recognizes a selected antigen.
In another aspect, the invention features, a cell, e.g., a hematopoietic cell, e.g., a B lymphocyte, or, a clonal population or substantially purified preparation of such cells, preferably produced by a method of the invention described herein. Preferably, the cells misexpress Aiolos.
In another aspect, the invention features, a cell which produces or over produces an antibody, e.g., an IgA, IgG, or IgE antibody. The cell can be, e.g., a hematopoietic cell, e.g., a B lymphocyte, or a population, or substantially purified preparation, of such cells, preferably produced by a method of the invention described herein. Preferrably the cells misexpress Aiolos.
In another aspect, the invention features, a proliferation-deregulated cell. The cell can be, e.g., a hematopoietic cell, e.g., a B lymphocyte, or a population, or substantially purified preparation, of such cells, preferably produced by a method of the invention described herein.
In another aspect, the invention features, a lymphocyte, e.g., a B lymphoctye, or, a substantially homogenous population or substantially purified preparation of lymphocytes, preferably produced by a method of the invention described herein, which lymphocytes or population recognize a selected antigen. Preferably, the lymphocytes misexpress Aiolos.
In another aspect, the invention features, a method of culturing an Aiolos-misexpressing cell having at least one mutant allele at the Aiolos locus. The cell can be, e.g., a hematopoietic cell, e.g., a B lymphocyte. The method includes: introducing the cell into a mammal, wherein, preferably, the mammal is other than the one from which the cell has been isolated originally; and culturing the cell.
In a preferred embodiment, the method further includes: allowing the cell to proliferate in the mammal.
In preferred embodiments: the mammal is a non-human mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse.
In a preferred embodiment, the method further includes: allowing the Aiolos-misexpressing cell cell to divide and give rise to a proliferation-deregulated cell , e.g., a transformed lymphocyte; providing a plurality of the proliferation-deregulated cells e.g., lymphocytes or transformed lymphocytes from the mammal.
In preferred embodiments: the mammal, the cell or both, are heterozygous at the Aiolos locus; the mammal, the cell or both, carry a mutation at the Aiolos gene, e.g., a point mutation in or a deletion for all or part of the Aiolos gene, e.g., a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediates DNA binding of the Aiolos protein or for one or more of the two C terminal zinc finger regions which mediate dimerization of the Aiolos protein; the mammal is heterozygous or homozygous for an Aiolos transgene;the mammal, the cell or both, carry a mutation in the control region of the Aiolos gene.
In preferred embodiments: the mammal, the cell or both, carry a mutation at the Aiolos gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the mammal, the cell or both, carry a deletion for all or part of exon 7.
In preferred embodiments: the Aiolos-misexpressing cell is a homozygous mutant Aiolos cell e.g., a lymphocyte; the Aiolos-misexpressing cell is a B lymphocyte; the Aiolos-misexpressing cell is heterozygous or homozygous for an Aiolos transgene.
In preferred embodiments, the Aiolos-misexpressing cell is a lymphocyte and is: a cell which secretes one or more anti-inflammatory cytokines; a cell which is antigen or idiotype specific; a cell which produces, or over produces, antibodies, e.g., IgG, IgA, or IgE antibodies.
In preferred embodiments: the mammal is immunized with an antigen; the cell is exogenously supplied and one or both of the mammal or the mammal which donates the cell are immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen; an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In a preferred embodiment: the Aiolos-misexpressing cell, e.g., a lymphocyte, is supplied exogenously to the mammal, e.g., to a homozygous wild-type Aiolos mammal or a mammal carrying a mutation at the Aiolos gene, e.g., a point mutation or a deletion for all or part of the Aiolos gene. If exogenously supplied, the cell can be a human or a nonhuman, e.g., a swine, nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, lymphocyte.
Aiolos wild type cells can be cultured in Aiolos misexppressing mammals.
In another aspect, the invention features, a method of modulating the activity of, or promoting the interaction of an Aiolos misexpressing cell with, a target tissue or cell. The. method includes: supplying the target; and exposing the target to a Aiolos misexpressing cell, e.g., a hematopoietic cell, e.g., a B lymphocyte, preferably having at least one mutant allele at the Aiolos locus, preferably provided that: the target is not Aiolos-misexpressing; the target and the cell differ in genotype at a locus other than the Aiolos locus; the target and the cell are from different animals; the target and the cell are from different species; the target activity is modulated in a recipient mammal and either the target or the cell is from a donor mammal other than the recipient mammal; or the target is exposed to the cell in an in vitro system.
In a preferred embodiment: the donor of the Aiolos-misexpressing cell is heterozygous or homozygous for an Aiolos transgene; the donor of the Aiolos-misexpressing cell is heterozygous at the Aiolos locus; the donor of the Aiolos-misexpressing cell carries a point mutation in or a deletion for all or part of the Aiolos gene, e.g., mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediate Aiolos binding to DNA or in one or both of the C-terminal zinc finger regions which mediates Aiolos dimerization; the donor of the Aiolos-misexpressing cell is human or a non-human mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a rat or a mouse. In preferred embodiments, e.g., in the case of the human donor, the manipulation that gives rise to Aiolos deregulation, e.g., an Aiolos lesion, can be made in vitro.
In preferred embodiments: the mammal which provides the Aiolos misexpressing cell carries a mutation at the Aiolos gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the mammal carries deletion for all or part of exon 7.
In another preferred embodiment: the cell is heterozygous or homozygous for an Aiolos transgene; the cell is a heterozygous Aiolos cell; the cell is a homozygous mutant Aiolos cell; the lymphocyte is a B lymphocyte.
In preferred embodiments, the cell is a lymphocyte and is: a B cell; a cell which secretes one or more anti-inflammatory cytokines; a T cell which is antigen or idiotype specific.
In a preferred embodiment: the method is performed in an in vitro system; the method is performed in vivo, e.g., in a mammal, e.g., a rodent, e.g., a mouse or a rat, or a primate, e.g., a non-human primate or a human. If the method is performed in vitro, the donor of the target cell or tissue and the lymphocyte can be same or different. If the method is performed in vivo, there is a recipient animal and one or more donors.
In preferred embodiments: the method is performed in vivo and one or more of the recipient, the donor of the target cell or tissue, the donor of the cell, is immunized with an antigen; the method is performed in vitro and one or more of the donor of the target cell or tissue, the donor of the cell is immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen or an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In a preferred embodiment: the target is selected from a group consisting of T or B lymphocytes, macrophages, inflammatory leukocytes, e.g., neutrophils or eosinophils, mononuclear phagocytes, NK cells or T lymphocytes; the target is an antigen presenting cell, e.g., a professional antigen presenting cell or a nonprofessional antigen presenting cell; the target is spleen tissue, bone marrow tissue, lymph node tissue or thymic tissue, or the target is a syngeneic, allogeneic, or xenogeneic tissue.
In another preferred embodiment, the target is from a mammal, e.g., a human; the mammal is a non-human mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a rat or a mouse.
In preferred embodiments, the activity of the target which is modulated is: the production of a cytokine; the proliferation or activation of a cell of the immune system; the production of an antibody; the lysis of an antigen presenting cell or the activation of a cytolytic T lymphocyte; the effect of target on resistance to infection; the effect of target on life span; the effect of target on body weight; the effect of target on the presence, function, or morphology of tissues or organs of the immune system; the effect of target on the ability of a component of the immune system to respond to a stimulus (e.g., a diffusable substance, e.g., cytokines, other cells of the immune system, or antigens); the effect of target on the ability to exhibit immunological tolerance to an alloantigen or a xenoantigen.
In preferred embodiments the interaction is the binding of an antibody produced by the Aiolos misexpressing cell with the target.
In preferred embodiments: the target and the cell differ in genotype at a locus other than the Aiolos locus; the target and the cell are from different animals; the target is not Aiolos-misexpressing.
In another aspect, the invention features, a method of reconstituting an immune system. The method includes: supplying a recipient mammal, and introducing, preferably exogenously, into the recipient mammal, an immune system component from a donor mammal, which is Aiolos misexpressing, e.g., which carries at least one mutant allele at the Aiolos locus. The recipient mammal, can be, e.g., a human or a nonhuman mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse. The donor mammal can be, e.g., a human or a nonhuman mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a rat or a mouse. If the donor mammal is human, the manipulation that gives rise to Aiolos misexpression e.g., an the introduction of an Aiolos lesion, can be made in vitro. The donor mammal and the recipient mammal can be different individuals or the same individual.
In preferred embodiments, the component is or includes an Aiolos misexpressing cell, e.g., a hematopoietic cell, e.g., a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte.
In preferred embodiments, the component is from a donor mammal, e.g., a human or a nonhuman mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a rat or a mouse.
In a preferred embodiment, the method further includes: prior to introduction of a component into the subject, treating the lymphocyte to inhibit proliferation, e.g., by irradiating the component.
In a preferred embodiment, the donor mammal carries a mutation at the Aiolos gene, e.g., a deletion of all or part of the Aiolos gene.
In another preferred embodiment: the immune system component is any of a T cell, a T cell progenitor, a totipotent hematopoietic stem cell, a pluripotent hematopoietic stem cell, a B cell, a B cell progenitor, a natural killer cell, a natural killer cell progenitor, bone marrow tissue, spleen tissue, or thymic tissue.
In a preferred embodiment: the immune system component is from the same species as the recipient mammal; the immune system component is from species different from the species of the recipient mammal.
In preferred embodiments: the recipient mammal is a wild-type animal; an animal model for a human disease, e.g., a NOD mouse; the animal is immunocompromised by irradiation, chemotherapy, or genetic defect, e.g., the animal is a SCID mouse or a nude mouse; the recipient is deficient in an immune function, e.g., the recipient has been thymectomized, depleted of an immune system component, e.g., of cells or antibodies; the recipient has been administered chemotherapy or irradiation.
In preferred embodiments: the immune system component is heterozygous at the Aiolos locus; the immune system component is carries a mutation at the-Aiolos gene, e.g., a point mutation in or a deletion for all or part of the Aiolos gene, e.g., a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediates DNA binding of the Aiolos protein or for one or more of the two C terminal zinc finger regions which mediate dimerization of the Aiolos protein;the immune system component is heterozygous or homozygous for an Aiolos transgene; the immune system component carries a mutation in the control region of the Aiolos gene.
In preferred embodiments: the immune system component carries a mutation at the Aiolos gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the immune system component carries deletion for all or part of exon 7.
In preferred embodiments: the method is performed in vivo, and the recipient mammal or the donor mammal or both are immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen or an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In a preferred embodiment, the method further includes: determining a value for a parameter related to immune system function. The parameter related to the immune system function can be any of: the production of a cytokine; the proliferation or activation of a cell of the immune system; the production of an antibody; the lysis of an antigen presenting cell or the activation of a cytolytic T lymphocyte; resistance to infection; life span; body weight; the presence, function, or morphology of tissues or organs of the immune system; the ability of a component of the immune system to respond to a stimulus (e.g., a diffusable substance, e.g., cytokines, other cells of the immune system, or antigens); the ability to present an antigen; the ability to exhibit immunological tolerance to an alloantigen or a xenoantigen.
In another aspect, the invention features, a method of evaluating the interaction of an Aiolos misexpressing cell, e.g., a hematopoietic cell, a B lymphocyte, with an immune system component. The method includes: supplying an animal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse; introducing the cell and the immune component into the animal; and evaluating the interaction between the Aiolos misexpressing cell and the immune system component.
In a preferred embodiment, the method further includes: prior to introduction of a cell into the subject, treating the lymphocyte to inhibit proliferation, e.g., by irradiating the cell.
In a preferred embodiment: the immune system component is any of a T cell, a T cell progenitor, a totipotent hematopoietic stem cell, a pluripotent hematopojetic stem cell, a B cell, a B cell progenitor, a natural killer cell, a natural killer cell progenitor, bone marrow tissue, spleen tissue, or thymic tissue; the immune system component is from the same species as the animal; the immune system component is from species different from the species of the animal; the immune system component is from the same species as the lymphocyte; the immune system component is from species different from the species from which the lymphocyte is obtained.
In another preferred embodiment: the cell is from the same species as the animal; the cell is from a species which is different from the species of the animal.
In another preferred embodiment: the recipient mammal is a wild-type animal; an animal model for a human disease, e.g., a NOD mouse; the animal is immunocompromised by irradiation, chemotherapy, or genetic defect, e.g., the animal is a SCID mouse or a nude mouse; the recipient is deficient in an immune function, e.g., the recipient has been thymectomized, depleted of an immune system component, e.g., of cells or antibodies; the recipient has been administered chemotherapy or irradiation.
In a preferred embodiment: the cell is heterozygous or homozygous for an Aiolos transgene.
In preferred embodiments evaluating can include evaluating any of: the production of a cytokine; the proliferation or activation of a cell of the immune system; the production of an antibody; the lysis of an antigen presenting cell or the activation of a cytolytic T lymphocyte; resistance to infection; life span; body weight; the presence, function, or morphology of tissues or organs of the immune system; the ability of a component of the immune system to respond to a stimulus (e.g., a diffusable substance, e.g., cytokines, other cells of the immune system, or antigens); the ability to present an antigen; the ability to exhibit immunological tolerance to an alloantigen or a xenoantigen.
In preferred embodiments: the method is performed in vivo, and one or more of the animal, the donor of the Aiolos misexpressing cell, the donor of the immune system component, is immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen or an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In another aspect, the invention features, a mammal, e.g., a nonhuman mammal, e.g., e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, having an exogenously introduced immune system component, the component being from a human or nonhuman mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, or cell culture which is Aiolos misexpressing or which carries at least one mutant allele at the Aiolos locus. In preferred embodiments, e.g., if the immune system component is from a wild-type animal, e.g., a human, the manipulation that gives rise to Aiolos deregulation, e.g., an Aiolos lesion, can be made in vitro.
In preferred embodiments, the component is from a human or nonhuman mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, which is Aiolos misexpressing.
In preferred embodiments: the component is from a mammal which is Aiolos misexpressing; the component is from a mammal which is heterozygous at the Aiolos locus; the component is from a mammal which carries a mutation at the Aiolos gene, e.g., a point mutation in or a deletion for all or part of the Aiolos gene, e.g., a mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediates DNA binding of the Aiolos protein or for one or more of the two C terminal zinc finger regions which mediate dimerization of the Aiolos protein; the component is from a mammal which is heterozygous or homozygous for an Aiolos transgene; the component is from a mammal which carries a mutation in the control region of the Aiolos gene.
In preferred embodiments: the component is from a mammal which carries a mutation at the Aiolos gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the component is from a mammal which carries deletion for all or part of exon 7.
In preferred embodiments, the immune system component is: a helper T cell; cytolytic T cell; a suppressor T cell; a T cell which secretes one or more anti-inflammatory cytokines, e.g., IL-4, IL-10, or IL-13; a T cell which is antigen or idiotype specific; a suppressor T cell which is anti-idiotypic for an auto antibody or for an antibody which recognizes an allograft or xenograft tissue; the lymphocyte is an antigen-nonspecific T cell.
In another preferred embodiment: the immune system component is any of a T cell progenitor, a totipotent hematopoietic stem cell, a pluripotent hematopoietic stem cell, a B cell, a B cell progenitor, a natural killer cell, a natural killer cell progenitor, bone marrow tissue, spleen tissue, or thymic tissue; the immune system component is from the same species as the animal; the immune system component is from species different from the species of the animal.
In preferred embodiments: the mammal or the donor animal which produces the immune system component or both are immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen or an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In another aspect, the invention features, a reaction mixture, preferably an in vitro reaction mixture, including an immune system component, the component including cells which misexpress Aiolos or being from an animal or cell culture which is misexpresses Aiolos or which carries at least one mutant allele at the Aiolos locus, and a target tissue or cell, wherein preferably, the immune system component and the target differ in genotype at a locus other than the Aiolos or Ikaros locus; the component and the target are from different species, or the component and the target are from different animals.
In preferred embodiments, the component is from an animal or cell culture which misexpresses Aiolos.
In preferred embodiments: the immune system component is a lymphocyte heterozygous or homozygous for an Aiolos transgene, e.g., a transgene having a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains; the immune system component is a lymphocyte heterozygous or homozygous for a C terminal deletion.
In preferred embodiments, the immune system component is: a B cell.
In another preferred embodiment: the immune system component is any of a T cell progenitor, a totipotent hematopoietic stem cell, a pluripotent hematopoietic stem cell, a B cell, a B cell progenitor, a natural killer cell, a natural killer cell progenitor, bone marrow tissue, spleen tissue, or thymic tissue; the immune system component is from the same species as the target cell; the immune system component is from species different from the species of the target cell.
In a preferred embodiment: the target is selected from a group consisting of T or B lymphocytes, macrophages, inflammatory leukocytes, e.g., neutrophils or eosinophils, mononuclear phagocytes, NK cells or T lymphocytes; the target is an antigen presenting cell, e.g., a professional antigen presenting cell or a nonprofessional antigen presenting cell; the target is spleen tissue, lymph node tissue, bone marrow tissue or thymic tissue, or is syngeneic, allogeneic, xenogeneic, or congenic tissue.
In preferred embodiments: the donor of the immune system component or the donor of the target or both are immunized with an antigen. The antigen can be: an alloantigen; a xenoantigen or an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
In preferred embodiments the donor of the components is: a human or nonhuman mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or mouse. In preferred embodiments, e.g., in the case of a wild-type donor, e.g., a human, the manipulation that gives rise to Aiolos deregulation, e.g., an Aiolos lesion, can be introduced in vitro.
In preferred embodiments the donor of the target is: a human or nonhuman mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or mouse.
In preferred embodiments the reaction mixture includes an exogenously add cytokine or antigen, e.g., a protein antigen.
In another aspect, the invention features, a method of promoting or inhibiting the proliferation of a cell, or of modulating the entry of a cell into the cell cycle. The method includes: administering to the cell a compound which inhibits the formation Aiolos-Aiolos or Aiolos-Ikaros dimers. The method can be performed in vivo or in vitro. The cell can be, e.g., a hematopoietic cell, e.g., a B lymphocyte.
In preferred embodiments, the compound is: a competitive or noncompetitive inhibitor of the association of Aiolos or Ikaros subunits, e.g., a mutant Aiolos peptide, e.g., a mutant Aiolos peptide which has a mutation which inhibits the ability of the Aiolos protein to bind DNA but which does not inhibit the ability of the protein to form a dimer, e.g., a mutation in one or more of the four N terminal Zinc fingers binding regions. Aiolos mutants which have mutations which inhibit dimerization, e.g., mutations inone of more of the two C terminal zinc finger regions can also be used.
In preferred embodiments the compound is: a protein or peptide; a peptomimetic, a small molecule; a nucleic acid which encodes an inhibitor.
Methods for increasing cell division can be combined with procedures where it is desirable to increase cell division, e.g., the treatment, e.g., by chemotherapy or radiotherapy, of tumors or other cell-proliferative disorders.
Proliferation can be inhibited by administering wildtype Aiolos.
In another aspect, the invention features a cell, or purified preparation of cells, which include an Aiolos transgene, or which otherwise misexpress an Aiolos gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include an Aiolos transgene, e.g., a heterologous form of an Aiolos gene, e.g., a gene derived from humans (in the case of a non-human cell). The Aiolos transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous Aiolos gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed Aiolos alleles or for use in drug screening.
Cells, e.g., stem cells, treated by the method of the invention can be introduced into mammals, e.g., humans, non-human primates, or other mammals, e.g., rodents. In preferred embodiments the treatment is performed ex vivo and: the cell is autologous, e.g., it is returned to the same individual from which it was derived; the cell is allogeneic, i.e., it is from the same species as the mammal to which it is administered; the cell is xenogeneic, i.e., it is from a different species from the mammal to which it is administered.
An Aiolos-deregulated cell is a cell which has a mutant or misexpressed Aiolos gene, e.g., an inactiviated Aiolos gene.
A hematopoietic cell, can be, e.g., stem cell, e.g., a totipotent or a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte, e.g. a B lymphocyte or a T lymphocyte.
A proliferation-deregulated cell, as used herein, refers to a cell with other than wild
An Aiolos misexpressing animal, as used herein, is an animal in which one or more, and preferably substantially all, of the cells misexpress Aiolos.
A mutation at the Aiolos locus, as used herein, includes any mutation which alters the expression, structure, or activity of the Aiolos gene or its gene product. These include point mutations in and in particular deletions of all or part of the Aiolos coding region or its control region.
An exogenously supplied cell, tissue, or cell product, e.g., a cytokine, as used herein, is a cell, tissue, or a cell product which is derived from an animal other than the one to which is supplied or administered. It can be from the same species or from different species than the animal to which it is supplied.
A clonal population of lymphocytes, as used herein, is a population of two or more lymphocytes which have one or more of the following properties: they share a common stem cell ancestor; they share a common pre-thymocyte or pre b cell ancestor; they share a common thymocyte ancestor; they share the same T cell receptor genomic rearrangement;
they share a common CD4+CD8+ ancestor; they share a common CD4+ ancestor; they share a common CD8+ ancestor; they share a common CD4xe2x88x92CD8xe2x88x92 ancestor; they recognize the same antigen.
A substantially homogenous population of two or more cells e.g., lymphocytes, as used herein, means a population of cells in which at least 50% of the cells, more preferably at least 70% of the cells, more preferably at least 80% of the cells, most preferably at least 90%, 95% or 99% of the subject cell type, e.g., lymphocytes. With respect to the Aiolos locus however, the cells can be all (+/xe2x88x92), all (xe2x88x92/xe2x88x92), or a mixture of (+/xe2x88x92) and (xe2x88x92/xe2x88x92) cells.
Culturing, as used herein, means contacting a cell or tissue with an environment which will support viability of the cell or tissue and which preferably supports proliferation of the cell or tissue.
A substantially purified preparation of cells, e.g., lymphocytes, as used herein, means a preparation of cells in which at least 50% of the cells, more preferably at least 70% of the cells, more preferably at least 80% of the cells, most preferably at least 90%, 95% or 99% of the cells of the subject cell, e.g., are lymphocytes. With respect to the Aiolos locus however, the cells can be all (+/xe2x88x92), all (xe2x88x92/xe2x88x92), or a mixture of (+/xe2x88x92) and (xe2x88x92/xe2x88x92) cells.
Immunocompromised, as used herein, refers to a mammal in which at least one aspect of the immune system functions below the levels observed in a wild-type mammal. The mammal can be immunocompromised by a chemical treatment, by irradiation, or by a genetic lesion resulting in, e.g., a nude, a beige, a nude-beige, or an Ikarosxe2x80x94phenotype. The mammal can also be immunocompromised by an acquired disorder, e.g., by a virus, e.g., HIV.
As used herein, an Aiolos transgene, is a transgene which includes all or part of an Aiolos coding sequence or regulatory sequence. The term also includes DNA sequences which when integrated into the genome disrupt or otherwise mutagenize the Aiolos locus. Aiolos transgenes sequences which when integrated result in a deletion of all or part of the Aiolos gene. Included are transgenes: which upon insertion result in the misexpression of an endogenous Aiolos gene; which upon insertion result in an additional copy of an Aiolos gene in the cell; which upon insertion place a non-Aiolos gene under the control of an Aiolos regulatory region. Also included are transgenes: which include a copy of the Aiolos gene having a mutation, e.g., a deletion or other mutation which results in misexpression of the transgene (as compared with wild type); which include a functional copy of an Aiolos gene (i.e., a sequence having at least 5% of a wild type activity, e.g., the ability to support the development of T, B, or NK cells); which include a functional (i.e., having at least 5% of a wild type activity, e.g., at least 5% of a wild type level of transcription) or nonfunctional (i.e., having less than 5% of a wild type activity, e.g., less than a 5% of a wild type level of transcription) Aiolos regulatory region which can (optionally) be operably linked to a nucleic acid sequence which encodes a wild type or mutant Aiolos gene product or, a gene product other than an Aiolos gene product, e.g., a reporter gene, a toxin gene, or a gene which is to be expressed in a tissue or at a developmental stage at which Aiolos is expressed. Preferably, the transgene includes at least 10, 20, 30, 40 , 50, 100, 200, 500, 1,000, or 2,000 base pairs which have at least 50, 60, 70, 80, 90, 95, or 99% homology with a naturally occurring Aiolos sequence. Preferably, the transgene includes a deletion of all or some of exons 3 and 4, or a deletion for some or all of exon 7 of the Aiolos gene.
A xe2x80x9cheterologous promoterxe2x80x9d,.as used herein is a promoter which is not naturally associated with the Aiolos gene.
A xe2x80x9cpurified preparationxe2x80x9d or a xe2x80x9csubstantially pure preparationxe2x80x9d of an Aiolos polypeptide, or a fragment or analog thereof (or an Aiolos-Aiolos or Aiolos-Ikaros dimer), as used herein, means an Aiolos polypeptide, or a fragment or analog thereof (or an Aiolos-Aiolos or Aiolos-Ikaros dimer), which is free of one or more other proteins lipids, and nucleic acids with which the Aiolos polypeptide (or an Aiolos-Aiolos or Aiolos-Ikaros dimer) naturally occurs. Preferably, the polypeptide, or a fragment or analog thereof (or an Aiolos-Aiolos or Aiolos-Ikaros dimer), is also separated from substances which are used to purify it, e.g., antibodies or gel matrix, such as polyacrylamide. Preferably, the polypeptide, or a fragment or analog thereof (or an Aiolos-Aiolos or Aiolos-Ikaros dimer), constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 xcexcg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
A xe2x80x9csubstantially pure nucleic acidxe2x80x9d, e.g., a substantially pure DNA encoding an Aiolos polypeptide, is a nucleic acid which is one or both of: not immediately contiguous with one or both of the coding sequences with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional Aiolos sequences.
xe2x80x9cHomologousxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions comparedxc3x97100. For example, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., one or more Aiolos polypeptides or Aiolos-Ikaros dimers), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence, such as the Aiolos and/or Ikaros gene, operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as lymphocytes. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
A polypeptide has Aiolos biological activity if it has one or more of the following properties: (1) the ability to react with an antibody, or antibody fragment, specific for (a) a wild type Aiolos polypeptide, (b) a naturally-occurring mutant Aiolos polypeptide, or (c) a fragment of either (a) or (b); (2) the ability to form Aiolos dimers and/or Aiolos/Ikaros dimers; (3) the ability to modulate lymphocyte differentiation; (4) the ability to stimulate transcription from a sequence, e.g., a sequence described herein; or (5) the ability to act as an antagonist or agonist of the activities recited in (1), (2), (3) or (4).
xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of Aiolos gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing, size, amino acid sequence, post-transitional modification, stability, or biological activity of the expressed Aiolos and/or Ikaros polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the Aiolos and/or Ikaros gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus; a ratio of Ikaros-Ikaros dimer to Aiolos-Aiolos dimer which differs from wild type; a ratio of Aiolos to Aiolos-Aiolos dimer, Ikaros-Ikaros dimer, or Ikaros-Aiolos dimer that differs from wild type; a ratio of Ikaros-Aiolos dimer to Aiolos, Ikaros, Aiolos-Aiolos dimer, or Ikaros-Ikaros dimer that differs from wild type.
As described herein, one aspect of the invention features a pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding an Aiolos, and/or equivalents of such nucleic acids. The term xe2x80x9cnucleic acidxe2x80x9d, as used herein, can include fragments and equivalents. The term xe2x80x9cequivalentxe2x80x9d refers to nucleotide sequences encoding functionally equivalent polypeptides or functionally equivalent polypeptides which, for example, retain the ability to react with an antibody specific for an Aiolos polypeptide. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and will, therefore, include sequences that differ from the nucleotide sequence of Aiolos shown in SEQ ID NO:1 or SEQ ID NO:7 due to the degeneracy of the genetic code.
An Aiolos-responsive control element, as used herein is a region of DNA which, when present upstream or downstream from a gene, results in regulation, e.g., increased transcription of the gene in the presence of an Aiolos protein.
A peptide has Ikaros activity if it has one or more of the following properties: the ability to stimulate transcription of a DNA sequence under the control any of a xcex4A element, an NFKB element, or one of the Ikaros binding oligonucleotide consensus sequences disclosed herein; the ability to bind to any of a xcex4A element, an NFKB element, or one of the Ikaros binding oligonucleotide consensus sequences disclosed herein; or the ability to competitively inhibit the binding of a naturally occurring Ikaros isoform to any of a xcex4A element, an NFKB element, or one of the Ikaros binding oligonucleotide consensus sequences disclosed herein. An Ikaros peptide is a peptide with Ikaros activity.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
The Aiolos genes and polypeptides of the present invention are useful for studying, diagnosing and/or treating diseases associated with unwanted cell proliferation, e.g., leukemias or lymphomas. The gene (or fragment thereof) can be used to prepare antisense constructs capable of inhibiting expression of a mutant or wild type Aiolos gene encoding a polypeptide having an undesirable function. Alternatively, an Aiolos polypeptide can be used to raise antibodies capable of detecting proteins or protein levels associated with abnormal cell proliferation or lymphocyte differentiation, e.g., T cell maturation. Furthermore, Aiolos peptides, antibodies or nucleic acids, can be used to identify the stage of lymphocyte differentiation, e.g., the stage of T cell differntiation.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.